Single Primer Amplification Research Articles

Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.

The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil, November 2019.

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log10 SARS-CoV-2 genome copies (GC) L−1), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log10 SARS-CoV-2 GC L−1, ranging from 5.49 ± 0.02 log10 GC L−1 (27th November 2019) to 6.68 ± 0.02 log10 GC L−1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

Genetic Diversity Analysis, Population Structure and Gene-flow Estimates among Citrus Macroptera Mont. Collections from Garo Hills, Meghalaya using SPAR Approach

North East India is extremely rich in natural resources and is a biodiversity hotspot. Many plant species, including Citrus are known to have originated from this region. Citus macroptera Mont. is a wild, endangered species which have been found to exist naturally in various parts of this region, including Meghalaya. To access the genetic variability among genotypes and their phylogeny, 30 genotypes of wild C. macroptera Mont. were collected from Garo Hills of Meghalaya. Single primer based DNA markers viz. RAPD, ISSR, DAMD were utilized to ascertain genetic diversity. The percentage polymorphic bands for RAPD, ISSR, DAMD were found to be 97.71%, 94.67% and 100% respectively. ISSR showed the highest values for both RP (7.67) and MI (5.03) highlighting its efficacy in determining genetic variations. A concatenated approach, Single Primer Amplification Reaction (SPAR) was also followed to assess their genetic diversity. Dendogram generated from SPAR data showed that the South-West Garo Hills population is the most recently evolved amongst all others while West Garo Hills collections retain an ancestral position in the evolutionary time-frame. Population genetics parametres such as Gene flow (Nm) and the diversity among populations (GST) were found to be 1.9894 and 0.2009 respectively. Gene flow estimates (Nm>1) suggests appreciable gene flow in the populations. AMOVA data further supported this with high percentage of variations (92%) within populations whereas variations among populations were about 8% only. Shannon’s information index (I) values and Nei’s gene diversity (h) varied between 0.303-0.423and 0.201-0.285 respectively. The use of SPAR method yields a clear and concise picture of the underlying genetic variabilities, and a detailed and comprehensive data analysis will help conceive efficient and sustainable conservation strategies for this important plant.

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from a Fish Farm in Scotland.

The aquatic virus, infectious pancreatic necrosis virus (IPNV), is known to infect various farmed fish, in particular salmonids, and is responsible for large economic losses in the aquaculture industry. Common practices to detect the virus include qPCR tests based on specific primers and serum neutralization tests for virus serotyping. Following the potential presence of IPNV viruses in a fish farm in Scotland containing vaccinated and IPNV-resistant fish, the common serotyping of the IPNV isolates was not made possible. This led us to determine the complete genome of the new IPNV isolates in order to investigate the cause of the serotyping discrepancy. Next-generation sequencing using the Illumina technology along with the sequence-independent single primer amplification (SISPA) approach was conducted to fully characterize the new Scottish isolates. With this approach, the full genome of two isolates, V1810–4 and V1810–6, was determined and analyzed. The potential origin of the virus isolates was investigated by phylogenetic analyses along with tridimensional and secondary protein structure analyses. These revealed the emergence of a new variant from one of the main virus serotypes, probably caused by the presence of selective pressure exerted by the vaccinated IPNV-resistant farmed fish.

Genetic Diversity of Rift Valley Fever Strains Circulating in Namibia in 2010 and 2011.

Outbreaks of Rift Valley fever (RVF) occurred in Namibia in 2010 and 2011. Complete genome characterization was obtained from virus isolates collected during disease outbreaks in southern Namibia in 2010 and from wildlife in Etosha National Park in 2011, close to the area where RVF outbreaks occurred in domestic livestock. The virus strains were sequenced using Sanger sequencing (Namibia_2010) or next generation sequencing (Namibia_2011). A sequence-independent, single-primer amplification (SISPA) protocol was used in combination with the Illumina Next 500 sequencer. Phylogenetic analysis of the sequences of the small (S), medium (M), and large (L) genome segments of RVF virus (RVFV) provided evidence that two distinct RVFV strains circulated in the country. The strain collected in Namibia in 2010 is genetically similar to RVFV strains circulating in South Africa in 2009 and 2010, confirming that the outbreaks reported in the southern part of Namibia in 2010 were caused by possible dissemination of the infection from South Africa. Isolates collected in 2011 were close to RVFV isolates from 2010 collected in humans in Sudan and which belong to the large lineage containing RVFV strains that caused an outbreak in 2006–2008 in eastern Africa. This investigation showed that the RVFV strains circulating in Namibia in 2010 and 2011 were from two different introductions and that RVFV has the ability to move across regions. This supports the need for risk-based surveillance and monitoring.

SPAR methods reveal high genetic diversity within populations and moderate gene flow of pointed gourd (Trichosanthes dioica Roxb.) germplasm

Abstract Pointed gourd (Trichosanthes dioica Roxb.) is one of the most nutritive cucurbitaceous vegetable crops cultivated in tropical and subtropical regions around the world. Poor characterization of the available germplasm, vegetative means of propagation and dioecy are the main constraints in developing a systematic breeding programme in pointed gourd. In this investigation, genetic variability of pointed gourd accessions belonging to four agro-climatic zones of Bengal region of India was evaluated by employing polymerase chain reaction (PCR) based single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and directed amplification of minisatellite DNA (DAMD). Percentage of polymorphic loci, Nei’s gene diversity index (h) and Shannon’s Information index (I) indicated a moderate level of genetic heterogeneity in studied germplasm. The mean coefficient of genetic differentiation (Gst) between populations for RAPD, ISSR and DAMD were 0.419, 0.305 and 0.315 respectively, indicating that 58.1%, 69.5% and 68.5% of genetic variability prevailed within the population. Similarly, analysis of molecular variance (AMOVA) revealed that majority of the genetic variation were partitioned among the individuals within a population. Gene flow estimate (Nm) calculated for RAPD, ISSR and DAMD marker were 0.692, 1.139 and 1.086 respectively indicating a moderate gene flow among the populations. The clustering pattern based on the combined marker systems was primarily related to the geographical distribution of the accessions. This study clearly demonstrates the efficiency of single primer based amplification reactions (SPARs) in duplicate identification, core collection and improvement of pointed gourd germplasm.

Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschussifanicus).

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD), and its infection results in mortality of 70–90% in farmed and wild rabbits. RHDV is thought to replicate strictly in rabbits. However, there are also reports showing that gene segments from the RHDV genome or antibodies against RHDV have been detected in other animals. Here, we report the detection and isolation of a RHDV from diseased Alpine musk deer (Moschus sifanicus). The clinical manifestations in those deer were sudden death without clinical signs and hemorrhage in the internal organs. To identify the potential causative agents of the disease, we used sequence independent single primer amplification (SISPA) to detect gene segments from viruses in the tissue samples collected from the dead deer. From the obtained sequences, we identified some gene fragments showing very high nucleotide sequence similarity with RHDV genome. Furthermore, we identified caliciviral particles using an electron microscope in the samples. The new virus was designated as RHDV GS/YZ. We then designed primers based on the genome sequence of an RHDV strain CD/China to amplify and sequence the whole genome of the virus. The genome of the virus was determined to be 7437 nucleotides in length, sharing the highest genome sequence identity of 98.7% with a Chinese rabbit strain HB. The virus was assigned to the G2 genotype of RHDVs according to the phylogenetic analyses based on both the full-length genome and VP60 gene sequences. Animal experiments showed that GS/YZ infection in rabbits resulted in the macroscopic and microscopic lesions similar to that caused by the other RHDVs. This is the first report of RHDV isolated from Alpine musk deer, and our findings extended the epidemiology and host range of RHDV.

Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the coronavirus disease 2019 (COVID‐19) pandemic. Accurate detection of SARS‐CoV‐2 using molecular assays is critical for patient management and the control of the COVID‐19 pandemic. However, there is an increasing number of SARS‐CoV‐2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence‐independent single‐primer amplification and nanopore whole‐genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5′ end of the SARS‐CoV‐2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID‐19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real‐time RT‐PCR assay. The primers and probes are highly specific for SARS‐CoV‐2. Validation with 101 clinical specimens showed that our nsp1 RT‐PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%‐97.2%), which was similar to those of N and E gene RT‐PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%‐100%). The addition of nsp1 for multitarget detection of SARS‐CoV‐2 can avoid false‐negative results due to mutations at the primers/probes binding sites of currently available RT‐PCR assays.

Molecular analyses of genetic variability in the populations of Bergenia ciliata in Indian Himalayan Region (IHR).

Bergenia ciliata is an important medicinal plant species of Indian Himalayan Region (IHR). Genetic variability and population genetic structure of B. ciliata sampled from IHR was studied using two single primer amplification reaction (SPAR) methods (DAMD: Directed Amplification of Minisatellite region DNA; ISSR: Inter Simple Sequence Repeats). To provide a reasonable scientific basis for management and conservation of B. ciliata populations in IHR, genetic diversity analysis of 11 populations with 24 SPAR markers (15 ISSR and 9 DAMD) revealed significantly high level of (90.03%) polymorphism at species level. However, genetic variability was low at population level and KUL and BWS populations showed maximum while SHM population revealed least genetic diversity among the 11 populations. Analysis of molecular variance revealed highest percentage of variation (73%) within populations, followed by 17% among populations and least (10%) among the Himalayan regions. Clustering pattern obtained from UPGMA dendrogram was supported by STRUCTURE and principal coordinate analysis, segregating all the 11 natural populations of B. ciliata into two genetic clusters: Eastern and Western Himalayan populations. The clustering patterns of all the three statistical methods indicated that populations of B. ciliata have structured in response to the local micro-climates of the habitats in IHR, and therefore, it can be concluded that genetic variability is in congruence with the geographical diversity.

Effects of sample processing modes on high-throughput sequencing of coronavirus whole genome

Objective To study the effects of different pre-sequencing sample processing modes on the results of whole genome sequencing with high-throughput sequencing (HTS) by taking the largest RNA virus (human coronavirus, HCoV) as the representative. Methods Cell-cultured human coronavirus HCoV-OC43 strains were used as the representative samples and divided into different groups based on pre-sequencing processing modes as follows: untreated group, DNase and RNase treatment before nucleic acid extraction group, DNase treatment after nucleic acid extraction group, and DNase and RNase treatment before nucleic acid extraction and DNase treatment after nucleic acid extraction group. Nucleic acid samples of each group were analyzed by direct RNA sequencing (without amplification) and DNA sequencing after sequence independent single primer amplification (SISPA), respectively. Results No significant difference in viral genome coverage rates was observed between different groups. The highest genome coverage and sequencing accuracy were obtained in DNase treatment after nucleic acid extraction group by direct RNA sequencing, and the ratio of viral reads and the sequencing depth of each locus were effectively improved by SISPA amplification. Conclusions This study provided an optimized technical strategy for whole genome sequencing of RNA viruses such as coronavirus. Key words: Human coronavirus; Sequence independent single primer amplification; Whole genome sequencing; High-throughput sequencing

First genomic characterization of a Belgian Enterovirus C104 using sequence-independent Nanopore sequencing

Because of the enormous variation in their genome sequence, genotyping enteroviruses by standard methods can prove to be quite challenging. Nanopore sequencing offers the potential to overcome the limitations of older techniques, but thus far, only amplicon-based strategies have been used to sequence complete enterovirus genomes. By combining a sequence-independent, single primer amplification (SISPA) for cDNA generation with next-generation sequencing using the Oxford Nanopore MinION, complete enterovirus genomes can be obtained in an easy-to-use, sequence-independent manner. To demonstrate its usability, we applied this technique to determine the complete genome sequence of an enterovirus C104 strain, representing the first documented occurrence of this uncommon enterovirus strain in Belgium.

Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

Augmenting a competent in vitro organogenesis etiquette from leaf base of country mallow, Abutilon indicum L. sweet: An ethno-botanically valuable medicinal plant

Abstract An easy, efficient and highly reproducible regeneration system was established through organogenesis from leaf base mediated callus of an ethnobotanical hairy shrub Abutilon indicum L. sweet, which is documented to possess pharmaceutically important phytochemicals. Among various explants, leaf bases produced significant callus induction (89%) with 140 mg fresh weight on Murashige and Skoog (MS) medium supplemented with 11.31 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D). Combinations of 2, 4-D and kinetin (KIN) increased the biomass (191 mg) of embryogenic calli. Regeneration medium with a combination of 8.88 μM of 6-amino benzyl purine (BAP) and 8.06 μM of naphthalene acetic acid in MS basal media significantly influenced the initiation of shoot primordium with 91% of regeneration. Proficiently regenerated shoot apical meristems (SAMs) produced higher frequency of multiple shoot induction on 4.10 μM of zeatin. The fully regenerated mature shootlets produced high root biomass (791.67 ± 48.05 mg) on half - strength MS with 4.54 μM indole-3-butyric acid (IBA) with an excellent mat root system for efficient acclimatization. In vitro established plantlets were successfully acclimatized under ex vitro conditions with high frequency. Presence of homologous DNA banding pattern with Single Primer Amplification Reaction markers confirmed the genetic similarity. FTIR and GC-MS spectrum showed similar metabolic profiles between wild and in vitro regenerated plantlets. Thus the protocol is reliable for in vitro regeneration through leaf base derived callus which could pave a way to the large scale production of secondary metabolite through biotechnological approaches.

Evaluating methods for Avian avulavirus-1 whole genome sequencing.

BackgroundAvian avulavirus-1 (AAvV-1, previously Newcastle Disease Virus) is responsible for poultry and wild birds' disease outbreaks. Numerous whole genome sequencing methods were reported for this virus. These methods included cloning, specific primers amplification, shotgun PCR approaches, Sequence Independent Single Primer Amplification and next generation sequencing platform kits.MethodsThree methods were used to sequence 173 Israeli Avian avulavirus-1 field isolates and one vaccine strain (VH). The sequencing was performed on Proton and Ion Torrent Personal Genome Machine and to a lesser extent, Illumina MiSeq and NextSeq sequencers. Target specific primers (SP) and Sequence Independent Single Primer Amplification (SISPA) products sequenced via the Ion torrent sequencer had a high error rate and truncated genomes. All the next generation sequencing platform sequencing kits generated high sequence accuracy and near-complete genomic size.ResultsA high level of mutations was observed in the intergenic regions between the avian avulavirus-1 genes. Within genes, multiple regions are more mutated than the Fusion region currently used for typing.ConclusionsOur findings suggest that the whole genome sequencing by the Ion torrent sequencing kit is sufficient. However, when higher fidelity is desired, the Illumina NextSeq and Proton torrent sequencing kits were found to be preferable.

Evaluation of bias induced by viral enrichment and random amplification protocols in metagenomic surveys of saliva DNA viruses

BackgroundViruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias.ResultsWe assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 μm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 μm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies.ConclusionsBenchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes.

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1–3 (HPIV1-3) were also obtained with the selected optimal methodology.

Genetic characterization of a novel picorna-like virus in Culex spp. mosquitoes from Mozambique

BackgroundMosquitoes are the potential vectors for a variety of viruses that can cause diseases in the human and animal populations. Viruses in the order Picornavirales infect a broad range of hosts, including mosquitoes. In this study, we aimed to characterize a novel picorna-like virus from the Culex spp. of mosquitoes from the Zambezi Valley of Mozambique.MethodsThe extracted RNA from mosquito pools was pre-amplified with the sequence independent single primer amplification (SISPA) method and subjected to high-throughput sequencing using the Ion Torrent platform. Reads that are classified as Iflaviridae, Picornaviridae and Dicistroviridae were assembled by CodonCode Aligner and SPAdes. Gaps between the viral contigs were sequenced by PCR. The genomic ends were analyzed by 5′ and 3′ RACE PCRs. The ORF was predicted with the NCBI ORF finder. The conserved domains were identified with ClustalW multiple sequence alignment, and a phylogenetic tree was built with MEGA. The presence of the virus in individual mosquito pools was detected by RT-PCR assay.ResultsA near full-length viral genome (9740 nt) was obtained in Culex mosquitoes that encoded a complete ORF (3112 aa), named Culex picorna-like virus (CuPV-1). The predicted ORF had 38% similarity to the Hubei picorna-like virus 35. The sequence of the conserved domains, Helicase-Protease-RNA-dependent RNA polymerase, were identified by multiple sequence alignment and found to be at the 3′ end, similar to iflaviruses. Phylogenetic analysis of the putative RdRP amino acid sequences indicated that the virus clustered with members of the Iflaviridae family. CuPV-1 was detected in both Culex and Mansonia individual pools with low infection rates.ConclusionsThe study reported a highly divergent, near full-length picorna-like virus genome from Culex spp. mosquitoes from Mozambique. The discovery and characterization of novel viruses in mosquitoes is an initial step, which will provide insights into mosquito-virus interaction mechanisms, genetic diversity and evolution.

Thidiazuron induced in vitro morphogenesis for sustainable supply of genetically true quality plantlets of Brahmi

An effective and rapid method for propagating genetically true plantlets of a pharmaceutically important plant Bacopa monnieri L. was developed from thidiazuron (TDZ) pulse treated nodal segment explants. Explants treated for 24 h with different concentration of 0.0, 5.0, 10, 20 or 40 μM thidiazuron (TDZ) prepared in liquid Murashige and Skoog (MS) medium induces multiple shoot on MS basal medium without growth regulators. The highest frequency 95.3 ± 2.3% of regeneration and maximum number 43.7 ± 2.0 of shoots per explants with an average shoot height of 5.4 ± 0.29 cm were achieved after 08 weeks of culture from the explants treated with 20 μM TDZ. Simultaneously, the regenerated microshoots were rooted on the same growth regulator free media. Elongated shoots with roots were successfully acclimated to ex vitro condition with a 97% survival rate. Genomic stability of in vitro developed plants of B. monnieri was first time determined using flow cytometry and single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA (RAPD), intersimple sequence repeat polymorphic DNA (ISSR) and directed amplification of minisatellite DNA (DAMD) and compared with donor plant. Nuclear DNA (nDNA) content of in vitro propagated B. monnieri plants has been estimated as 2.08 pg/2C based on the flow cytometric investigation of nuclei isolated from leaf tissues. No significant variations in nDNA content, plant genome size and ploidy level were observed between micropropagated plants and field grown donor plants. Likewise, the SPAR markers also revealed monomorphic banding profiles in micropropagated plantlets of B. monnieri and similar with to that of the donor plants verifying their genomic stability.

HLA class I (-A, -B, -C) and class II (-DR, -DQ) polymorphism in the Mauritanian population

BackgroundHLA antigens have been widely studied for their role in transplantation biology, human diseases and population diversity. The aim of this study was to provide the first profile of HLA class I and class II alleles in the Mauritanian population.MethodsHLA typing was carried in 93 healthy Mauritanian blood donors, using single specific primer amplification (PCR-SSP).ResultsOccurrences of the main HLA class I (-A, -B, -C) and class II (-DR, -DQ) antigens in the general population showed that out of the 17 HLA-A allele groups detected, five main HLA-A allele groups: A*02 (18.42%), A*01 (14.04%), A*23 (14.04%), A*30 (13.16%) and A*29 (12.28%) were the most common identified along other 12 relatively minor allele groups. Twenty three allele groups were observed in the locus B of which B*07 (13.46%) was the most prevalent followed by B*15, B*35, B*08 and B*27 all, with a frequency between 7 to 8%. Three prevalent HLA-C allele groups (C*02: 35.09%, C*07: 20.19% and C*06: 13.6%) were detected. The main HLA class II observed allele groups were: DRB1*13 (27.42%), DRB1*03 (24.73%), DRB1*11 (13.98%), DQB1*03 (36.03%), DQB1*02 (22.06%) and DQB1*05 (18.8%). Except for few haplotype in class I (A*02-B*07: 4.45%, A*02-C02: 10%, A*23-C*02: 8.8%, B*07-C*02: 8.8%, B*15-C*02: 8.8%) and in class II (DRB1*13-DQB1*06: 11.94%, DRB1*03-DQB1*02:11.19% and DRB1*03-DQB1*03: 10.45%), the majority of locus combination were in the range of 2–3%. A single predominant haplotype C*02-DRB1*03 (16.67%) was found.ConclusionsThese results, in agreement with previous data using different tissues markers, underlined the ethnic heterogeneity of the Mauritanian population.